CN108588278A - A kind of parrot beak ptilosis virus PCR diagnostic kit and its detection method - Google Patents
A kind of parrot beak ptilosis virus PCR diagnostic kit and its detection method Download PDFInfo
- Publication number
- CN108588278A CN108588278A CN201810359781.1A CN201810359781A CN108588278A CN 108588278 A CN108588278 A CN 108588278A CN 201810359781 A CN201810359781 A CN 201810359781A CN 108588278 A CN108588278 A CN 108588278A
- Authority
- CN
- China
- Prior art keywords
- pcr amplification
- reaction
- pcr
- pbfdv
- control sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of parrot beak ptilosis virus PCR diagnostic kit and its detection methods, including：Lysate, amplified reaction mixed liquor, negative control and positive control；Wherein, the ingredient of lysate is DNAiso Reagent；Amplified reaction mixed liquor includes sterilizing tri-distilled water, PCR Buffer, dNTP, PBFDV F sense primers, PBFDV R downstream primers and rTaq archaeal dna polymerases；Negative control is sterilizing tri-distilled water；Positive control is PBFDV positive plasmids.The kit devises a pair of of specific primer for parrot beak feather syndrome virus, the high sensitivity of kit, specificity is good, stability is high, visual result, its detection method is easily operated, convenient and efficient, detection time can substantially be shortened, reduce diagnosis expense, strong technological means is provided for the infection of clinic control parrot beak ptilosis virus, is suitable for the quick diagnosis of parrot beak feather syndrome virus disease.
Description
Technical field
The present invention relates to animal medicine animal epidemic diagnostic technique in molecular biology fields, and in particular to a kind of parrot beak feather
Sick virus PCR diagnostic kit and its detection method.
Background technology
The cause of disease of parrot beak feather disease is that parrot beak ptilosis is viral (PBFDV), mainly causes parrot beak and feather lesion, is
A kind of highly infective disease, morbidity is anxious, dead rapid.This disease finds the South Pacific Ocean parrot in 1970 mid-nineties 90s, disease first
Shape is that feather irregularly grows and falls off, beak deformation, finally dead because of immunity reduction initiation infection.Existed successively again later
Cockatoo, African African gray, compromise parrot, love parrot, budgerigar, Blue-fronted Amazon, macaw and small parrot hair
Existing PBFDV infection.
PBFDV belongs to circovirus section, Circovirus member, is that 20 face bodies are symmetrically viral, and genome is cyclic annular sub-thread
DNA.The approach that PBFDV is propagated mainly disseminates virus by blood, plumage bits, excrement, the crop secretion of suffering from bird, wherein and with
Plumage bits are most important distribution source, and birdling developing immune system is immature, so being easy to be infected.At present to PBFDV's
Diagnosis research is almost blank, and the present invention devises PCR diagnostic methods for PBFDV genes, and is assembled into kit, is parrot
PBFDV infects most sensitive special diagnostic techniques means.
Invention content
For problems of the prior art, the purpose of the present invention is to provide a kind of parrot beak ptilosis virus PCRs to examine
Disconnected kit and its detection method, the kit devise a pair of of specific primer for parrot beak feather syndrome virus, kit
High sensitivity, specificity is good, stability is high, visual result, and detection method is easily operated, convenient and efficient, can substantially shorten inspection
The time is surveyed, diagnosis expense is reduced, strong technological means is provided for the infection of clinic control parrot beak ptilosis virus, is suitable for parrot
The quick diagnosis of beak feather syndrome virus disease.
In order to achieve the above object, the present invention is achieved by the following scheme.
(1) a kind of parrot beak ptilosis virus PCR diagnostic kit, including：Lysate, amplified reaction mixed liquor, feminine gender are right
According to and positive control；Wherein, the ingredient of the lysate is DNAiso Reagent；The amplified reaction mixed liquor includes sterilizing
Tri-distilled water, PCR Buffer, dNTP, PBFDV-F sense primer, PBFDV-R downstream primers and rTaq archaeal dna polymerases；Described the moon
Property control for sterilizing tri-distilled water；The positive control is PBFDV positive plasmids.
Preferably, the sequence of the PBDFV-F sense primers is：5’-AGCGATTTGGATAATGAGAAGTA-3’；It is described
The sequence of PBDFV-R downstream primers is：5’-CCCGGGGGGCCGTACACAACGT-3’.
Preferably, the lysate ingredient is DNAiso Reagent, and 20 reactions amount to 20mL, dress up 1 bottle；The expansion
Increasing reaction mixture includes：Sterilize tri-distilled water, 10 × PCR Buffer, 2.5mmolL-1dNTP、25μmol·L-1PBFDV-F
Sense primer, 25 μm of olL-1PBFDV-R downstream primers and 5U/ μ L rTaq archaeal dna polymerases, each reaction volume ratio are
17.0 ︰, 2.5 ︰, 2 ︰, 0.5 ︰, 0.5 ︰ 0.5, amount to 23 μ L, and 20 reactions amount to 460 μ L, dress up 1 pipe；The negative control is sterilizing
Tri-distilled water, 20 reactions amount to 2.0mL, dress up 1 bottle；The positive control is PBFDV positive plasmids, 20 reactions total 40.0
μ L dress up 1 pipe.
(2) a kind of detection method of parrot beak ptilosis virus PCR diagnostic kit, includes the following steps：
Step 1, DNA is extracted
Sub-step 1.1, measuring samples DNA extractions：Measuring samples merging sterile centrifugation tube (EP pipes) is drawn, cracking is added
Liquid overturns mixing, stands cracking, and absolute ethyl alcohol is added, and overturns mixing, staticly settles, and centrifuges, discards supernatant liquid, washs, discards
Cleaning solution is inverted sterile centrifugation tube, spontaneously dries, dissolved with NaOH in air, obtain measuring samples DNA extracting solutions.
Sub-step 1.2, negative control sample extraction：It draws sterilizing tri-distilled water and is placed in sterile centrifugation tube, lysate, top is added
Mixing stands cracking, and absolute ethyl alcohol is added, and overturns mixing, staticly settles, and centrifuges, discards supernatant liquid, washs, discards washing
Liquid is inverted sterile centrifugation tube, spontaneously dries, dissolved with NaOH in air, obtain negative control sample extracting solution.
Step 2, pcr amplification reaction
Sub-step 2.1, measuring samples pcr amplification reaction：Amplified reaction mixed liquor is taken, the measuring samples DNA is added and carries
Take liquid, be uniformly mixed, carry out pcr amplification reaction, the condition of the pcr amplification reaction be followed successively by 95 DEG C of pre-degenerations reaction 5min,
94 DEG C of reaction 30s, 58 DEG C of reaction 40s, 72 DEG C are reacted 40s, carry out multiple cycles altogether, are finally reacted 10min at 72 DEG C again, must be waited for
Sample product pcr amplification product.
Sub-step 2.2, negative control sample pcr amplification reaction：Amplified reaction mixed liquor is taken, the negative control sample is added
Product extracting solution is uniformly mixed, and carries out pcr amplification reaction, and the condition of the pcr amplification reaction is followed successively by 95 DEG C of pre-degeneration reactions
5min, 94 DEG C of reactions 30s, 58 DEG C of reactions 40s, 72 DEG C of reaction 40s carry out multiple cycles, finally the reaction at 72 DEG C again altogether
10min obtains negative control sample pcr amplification product.
Sub-step 2.3, positive control sample pcr amplification reaction：Amplified reaction mixed liquor is taken, parrot beak ptilosis virus is added
Positive plasmid is uniformly mixed, and carries out pcr amplification reaction, and the condition of the pcr amplification reaction is followed successively by 95 DEG C of pre-degeneration reactions
5min, 94 DEG C of reactions 30s, 58 DEG C of reactions 40s, 72 DEG C of reaction 40s carry out multiple cycles, finally the reaction 10min at 72 DEG C altogether,
Obtain positive control sample pcr amplification product.
Step 3, electrophoresis detection
The measuring samples pcr amplification product, negative control sample PCR amplification are produced respectively by agarose gel electrophoresis
Object and positive control sample pcr amplification product carry out electrophoresis detection, and are judged.
Preferably, in step 2, the amplified reaction mixed liquor include sterilizing tri-distilled water, PCR Buffer, dNTP,
PBFDV-F sense primers, PBFDV-R downstream primers and rTaq archaeal dna polymerases.
Preferably, the sequence of the PBDFV-F sense primers is：5’-AGCGATTTGGATAATGAGAAGTA-3’；It is described
The sequence of PBDFV-R downstream primers is：5’-CCCGGGGGGCCGTACACAACGT-3’.
Preferably, in step 2, the multiple cycle is 35 cycles.
Preferably, in step 3, the standard of the judgement is：Negative control sample pcr amplification product not shaping band, it is positive
Control sample pcr amplification product goes out 260bp bands, illustrates that control is set up；Occur 260bp items in measuring samples pcr amplification product
Band, as parrot beak ptilosis virus infect；Measuring samples pcr amplification product is no band person, as negative.
Compared with prior art, beneficial effects of the present invention are：
The parrot beak ptilosis virus PCR diagnostic kit of gained of the invention can make a definite diagnosis whether parrot infects parrot in a short time
Nautilus beak ptilosis virus, at 2 hours or so, the specificity of the kit was good for detection time control, and energy specific diagnosis goes out parrot beak
Ptilosis virus；High sensitivity, detectable limit is up to 1.95 × 10-7pg·μL-1；Stability is high, and kit is being examined in 6 months
It is consistent to survey result；Field trial proves that the practicability of kit and applicability are good.Its detection method is easily operated, convenient fast
Victory can achieve the purpose that quickly to detect.
Description of the drawings
The present invention is described in further details in the following with reference to the drawings and specific embodiments.
Fig. 1 is the parrot beak ptilosis virus PCR diagnostic kit of the present invention and its specific test electrophoresis of detection method
Figure；
M is DL2000DNA molecular mass standards in figure, and 1 is fowlpox virus, and 2 be parrot beak ptilosis virus, and 3 be adenovirus,
4 be mycoplasma, and 5 be infectious laryngotracheitis virus, and 6 is viral for parrot beak ptilosis, and 7 be Escherichia coli Mycoplasma columbinum.
Fig. 2 is the parrot beak ptilosis virus PCR diagnostic kit of the present invention and its sensitivity test electrophoresis of detection method
Figure；
M is DL2000DNA molecular mass standards in figure, and 1~11 dilutes for parrot beak ptilosis viral sample DNA ladder degree, dense
Degree ranging from 195 × 10-1pg·μL-1~195 × 10-11pg·μL-1；
Fig. 3 is parrot beak ptilosis virus PCR diagnostic kit and its detection method of the invention to partial clinical sample
Testing result；
M is DL2000DNA molecular mass standards in figure, and P is positive control, and N is negative control；1-9 is part feather pulp, entirely
Blood and Virus monitory result.
Specific implementation mode
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
It will be appreciated that the following example is merely to illustrate the present invention, and it is not construed as limiting the scope of the invention.
Embodiment 1
A kind of parrot beak ptilosis virus PCR diagnostic kit, including：Lysate, amplified reaction mixed liquor, negative control and
Positive control, it is specific as follows：
1) lysate：Ingredient is DNAiso Reagent, and 20 reactions amount to 20mL, dress up 1 bottle.
2) amplified reaction mixed liquor：Sterilize tri-distilled water, 10 × PCR Buffer, 2.5mmolL-1dNTP、25μmol·L-1PBFDV-F sense primers, 25 μm of olL-1PBFDV-R downstream primers and 5U/ μ L rTaq archaeal dna polymerases, each reactant
Product amounts to 23 μ L, 20 reactions amount to 460 μ L, dress up 1 pipe than being 17.0 ︰, 2.5 ︰, 2 ︰, 0.5 ︰, 0.5 ︰ 0.5.
3) negative control：Sterilize tri-distilled water, and 20 reactions amount to 2.0mL, dress up 1 bottle.
4) positive control：PBFDV positive plasmids, 20 reactions amount to 40.0 μ L, dress up 1 pipe.
The wherein design of primer and preparation is as follows：
The sequence of PBDFV-F sense primers is：5’-AGCGATTTGGATAATGAGAAGTA-3’；PBDFV-R downstream primers
Sequence be：5’-CCCGGGGGGCCGTACACAACGT-3’；Above-mentioned primer is closed by Sangon Biotech (Shanghai) Co., Ltd.
At.
The detection method of above-mentioned parrot beak ptilosis virus PCR diagnostic kit, including following detecting step：
Step 1, DNA is extracted
Sub-step 1.1, measuring samples DNA extractions：100 μ L measuring samples merging 1.5mL sterile EP tubes are drawn, 800 μ are added
L lysates overturn mixing, stand cracking 10min, and the ice-cold absolute ethyl alcohols of 600 μ L are added, overturns mixing, staticly settles
10min, 12000r/min centrifuge 10min, discard supernatant liquid, are washed 1 time with 75% ethyl alcohol, discard ethyl alcohol, EP pipes are inverted, in sky
It is spontaneously dried in gas, with 40 μ L8mmolL-1NaOH dissolves, and obtains measuring samples DNA extracting solutions.
Sub-step 1.2, negative control sample extraction：100 μ L sterilizing tri-distilled water merging sterile centrifugation tubes are drawn, 800 μ are added
L lysates overturn mixing, stand cracking 10min, and the ice-cold absolute ethyl alcohols of 600 μ L are added, overturns mixing, staticly settles
10min, 12000r/min centrifuge 10min, discard supernatant liquid, are washed 1 time with 75% ethyl alcohol, discard ethyl alcohol, EP pipes are inverted, in sky
It is spontaneously dried in gas, with 40 μ L 8mmolL-1NaOH dissolves, and obtains negative control sample extracting solution.
Step 2, pcr amplification reaction
Sub-step 2.1, measuring samples pcr amplification reaction：23 μ L of amplified reaction mixed liquor are taken, measuring samples DNA is added and carries
2 μ L of liquid are taken, are uniformly mixed, carry out pcr amplification reaction, the condition of pcr amplification reaction is followed successively by 95 DEG C of pre-degenerations reaction 5min, and 94
DEG C reaction 30s, 58 DEG C reaction 40s, 72 DEG C reaction 40s, altogether carry out 35 cycle, finally at 72 DEG C react 10min, must wait for sample
Product pcr amplification product.
Sub-step 2.2, negative control sample pcr amplification reaction：23 μ L of amplified reaction mixed liquor are taken, negative control sample is added
2 μ L of product extracting solution are uniformly mixed, and carry out pcr amplification reaction, and the condition of pcr amplification reaction is followed successively by 95 DEG C of pre-degeneration reactions
5min, 94 DEG C of reaction 30s, 58 DEG C of reaction 40s, 72 DEG C of reaction 40s, 35 cycles of progress, finally react 10min at 72 DEG C altogether,
Obtain negative control sample pcr amplification product.
Sub-step 2.3, positive control sample pcr amplification reaction：23 μ L of amplified reaction mixed liquor are taken, parrot beak ptilosis is added
2 μ L of virus-positive plasmid are uniformly mixed, and carry out pcr amplification reaction, it is anti-that the condition of pcr amplification reaction is followed successively by 95 DEG C of pre-degenerations
5min is answered, 94 DEG C of reaction 30s, 58 DEG C of reaction 40s, 72 DEG C of reaction 40s, 35 cycles of progress, finally react at 72 DEG C altogether
10min obtains positive control sample pcr amplification product.
Step 3, electrophoresis detection
Pass through 10gL-1Agarose gel electrophoresis respectively expands measuring samples pcr amplification product, negative control sample PCR
Increase production object and positive control sample pcr amplification product carries out electrophoresis detection, and is judged；The standard of judgement is：Negative control
Not shaping band, positive control go out 260bp bands, illustrate that control is set up；Occur 260bp bands in measuring samples pcr amplification product,
As parrot beak ptilosis virus infects；Measuring samples pcr amplification product is no band person, as negative.
Lysate preservation condition is room temperature in the parrot beak ptilosis virus PCR diagnostic kit of the present invention, and amplified reaction is mixed
Close liquid, yin and yang attribute control preservation condition is -20 DEG C.
Specificity, the stabilization of parrot beak ptilosis virus PCR diagnostic kit and its detection method to 1 gained of embodiment
Property, sensitivity are tested, and the practicality as field trial to parrot beak ptilosis virus PCR diagnostic kit obtained by the present invention
Property and applicability are verified, specific as follows：
(1) specific test
Using the parrot beak ptilosis virus PCR diagnostic kit of the present invention, according to described above, fowl pox disease is extracted respectively
Poison, parrot beak ptilosis virus, adenovirus, mycoplasma, infectious laryngotracheitis virus, parrot beak ptilosis virus, e. coli dna
Template carries out PCR with the amplification mixed liquor of kit, electrophoresis observation after amplification, and test result is as shown in Figure 1.
As shown in Figure 1, there is mono- band of 260bp, fowlpox virus, parrot beak ptilosis virus, gland in parrot beak ptilosis virus
Virus, mycoplasma, infectious laryngotracheitis virus, Escherichia coli control do not occur band.The band of positive sample is recycled,
PMD18-T carriers are connected after purification, convert DH5 ɑ competent cells, PCR is taken to be accredited as positive bacterium solution, and extraction plasmid is surveyed
The sequence measured is compared by sequence with strain gene order used, it is found that sequence is completely the same.As a result the parrot of the present invention is confirmed
Nautilus beak ptilosis virus PCR diagnostic kit and detection method have very high specificity, can accurately expand parrot beak ptilosis virus
Genetic fragment.
(2) sensitivity test
Using parrot beak ptilosis virus PCR diagnostic kit provided by the invention, parrot beak ptilosis Virus Sample is extracted
DNA, ultraviolet specrophotometer measured concentration are 195pg μ L-1, 10 are diluted to according to 10 times of concentration gradients-11, carried with kit
The amplification mixed liquor progress PCR of confession, electrophoresis observation after amplification, test result are as shown in Figure 2.
As shown in Figure 2, the limit that parrot beak ptilosis virus PCR diagnostic kit of the invention can detect is 1.95 × 10- 7pg·μL-1, show that this method has very high sensitivity.
(3) stability test
Parrot beak ptilosis virus PCR diagnostic kit preservation condition provided by the invention is -20 DEG C, monthly uses parrot 1 time
Beak ptilosis virus and control are detected experiment, continuous detection 6 months, the stability of detection kit storage.The results show that 6
Kit testing result is completely the same in a month, illustrates that kit has good stability.
(4) field trial
Totally 95 parts of doubtful parrot beak feather disease case feather pulp, whole blood and serum are acquired, with the parrot beak ptilosis virus of the present invention
PCR diagnostic kits and detection method are diagnosed, specific as follows：
Embodiment 2
1, DNA is extracted
The processing of 1.1 feather pulp samples is extracted with DNA
Suspected case of choosing wing plumage 3-5 roots shred feather pulp, and 800 μ L lysates are added, and overturn mixing, stand cracking
10min, 4 DEG C, 12000r/min centrifugation 10min, draws 600 μ L supernatants and is placed in another centrifuge tube, it is ice-cold to add 600 μ L
Absolute ethyl alcohol overturns mixing, staticly settles 10min, and 4 DEG C, 12000r/min centrifugation 10min discard supernatant liquid, with 75% ethyl alcohol
Washing 1 time discards ethyl alcohol, is inverted in EP pipe air and spontaneously dries, with 40 μ L 8mmolL-1NaOH dissolves, and obtains feather pulp sample
DNA extracting solutions.
1.2 negative control samples extract：100 μ L sterilizing tri-distilled water merging sterile centrifugation tubes are drawn, 800 μ L cracking are added
Liquid overturns mixing, stands cracking 10min, and the ice-cold absolute ethyl alcohols of 600 μ L are added, overturns mixing, staticly settles 10min,
12000r/min centrifuges 10min, discards supernatant liquid, is washed 1 time with 75% ethyl alcohol, discards ethyl alcohol, is inverted EP pipes, in air certainly
It is so dry, with 40 μ L 8mmolL-1NaOH dissolves, and obtains negative control sample extracting solution.
2, pcr amplification reaction
2.1 feather pulp sample P CR amplified reactions：23 μ L of amplified reaction mixed liquor are taken, 2 μ L of feather pulp sample DNA extracting solution are added,
It is uniformly mixed, carries out pcr amplification reaction, the condition of pcr amplification reaction is followed successively by 95 DEG C of pre-degeneration reaction 5min, 94 DEG C of reactions
30s, 58 DEG C of reactions 40s, 72 DEG C of reaction 40s carry out 35 cycles altogether, and finally the reaction 10min at 72 DEG C, obtains feather pulp sample P CR
Amplified production.
2.2 negative control sample pcr amplification reactions：23 μ L of amplified reaction mixed liquor are taken, negative control sample extraction is added
2 μ L of liquid are uniformly mixed, and carry out pcr amplification reaction, and the condition of pcr amplification reaction is followed successively by 95 DEG C of pre-degenerations reaction 5min, 94 DEG C
30s is reacted, 58 DEG C of reaction 40s, 72 DEG C of reaction 40s, 35 cycles of progress, 10min is finally reacted at 72 DEG C, obtains negative control altogether
Sample P CR amplified productions.
2.3 positive control sample pcr amplification reactions：23 μ L of amplified reaction mixed liquor are taken, parrot beak ptilosis virus sun is added
2 μ L of property grain are uniformly mixed, and carry out pcr amplification reaction, and the condition of pcr amplification reaction is followed successively by 95 DEG C of pre-degeneration reaction 5min,
94 DEG C of reaction 30s, 58 DEG C of reaction 40s, 72 DEG C of reaction 40s, 35 cycles of progress, 10min is finally reacted at 72 DEG C, is obtained positive altogether
Control sample pcr amplification product.
3, electrophoresis detection
The feather pulp sample P CR amplified productions of gained, negative control sample pcr amplification product, positive control sample PCR are expanded
Volume increase object passes through 10gL-1Agarose gel electrophoresis carries out inspection diagnosis, specific as follows：
3.1 weigh 1.0g agaroses, are added in 100mL 1 × TAE buffer solutions, heating and melting in micro-wave oven, and 5 μ L are added
(100mg/mL) ethidium bromide, mixing are poured into horizontal positioned gel disk, and offset plate thickness is 5mm, is extracted after solidification to be cooled
Gel is put into electrophoresis tank by comb, and 1 × TAE buffer solutions are added and flood glue surface, obtain agarose buffer solution.
3.2 take 5 μ L feather pulp sample P CR amplified productions, negative control sample pcr amplification product, positive control sample respectively
Pcr amplification product and agarose buffer solution mixing are added in well, while adding 5 μ L DL2000DNA molecular weight standards.
3.3 voltage 80V~100V or electric current 40mA~50mA, electrophoresis 30min.
3.4 take out gel, are placed in uv analyzer and observe, or observe photograph in merging gel imaging system.
3.5 using DL2000DNA molecular masses standard as reference, and shaping band, positive control do not go out 260bp items to negative control
Band illustrates that control is set up；Occur 260bp bands in measuring samples pcr amplification product, as parrot beak ptilosis virus infects；It waits for
Sample product pcr amplification product is no band person, as negative.
Embodiment 3
1, the processing of blood serum sample
Wing venous blood sampling is carried out to suspected case parrot, natural coagulation detaches serum after serum precipitation, spare.
2, DNA is extracted
2.1 blood serum sample DNA extractions：Supernatant merging 1.5mL sterile EP tubes obtained by 100 μ L are drawn, 800 μ L cracking are added
Liquid overturns mixing, stands cracking 10min, and the ice-cold absolute ethyl alcohols of 600 μ L are added, overturns mixing, staticly settles 10min,
12000r/min centrifuges 10min, discards supernatant liquid, is washed 1 time with 75% ethyl alcohol, discards ethyl alcohol, is inverted EP pipes, in air certainly
It is so dry, with 40 μ L 8mmolL-1NaOH dissolves, and obtains blood serum sample DNA extracting solutions.
2.2 negative control samples extract：100 μ L sterilizing tri-distilled water merging sterile centrifugation tubes are drawn, 800 μ L cracking are added
Liquid overturns mixing, stands cracking 10min, and the ice-cold absolute ethyl alcohols of 600 μ L are added, overturns mixing, staticly settles 10min,
12000r/min centrifuges 10min, discards supernatant liquid, is washed 1 time with 75% ethyl alcohol, discards ethyl alcohol, is inverted EP pipes, in air certainly
It is so dry, with 40 μ L 8mmolL-1NaOH dissolves, and obtains negative control sample extracting solution.
3, pcr amplification reaction
3.1 blood serum sample pcr amplification reactions：23 μ L of amplified reaction mixed liquor are taken, 2 μ L of blood serum sample DNA extracting solutions are added,
It is uniformly mixed, carries out pcr amplification reaction, the condition of pcr amplification reaction is followed successively by 95 DEG C of pre-degeneration reaction 5min, 94 DEG C of reactions
30s, 58 DEG C of reactions 40s, 72 DEG C of reaction 40s carry out 35 cycles altogether, and finally the reaction 10min at 72 DEG C, obtains blood serum sample PCR
Amplified production.
3.2 negative control sample pcr amplification reactions：23 μ L of amplified reaction mixed liquor are taken, negative control sample extraction is added
2 μ L of liquid are uniformly mixed, and carry out pcr amplification reaction, and the condition of pcr amplification reaction is followed successively by 95 DEG C of pre-degenerations reaction 5min, 94 DEG C
30s is reacted, 58 DEG C of reaction 40s, 72 DEG C of reaction 40s, 35 cycles of progress, 10min is finally reacted at 72 DEG C, obtains negative control altogether
Sample P CR amplified productions.
3.3 positive control sample pcr amplification reactions：23 μ L of amplified reaction mixed liquor are taken, parrot beak ptilosis virus sun is added
2 μ L of property grain are uniformly mixed, and carry out pcr amplification reaction, and the condition of pcr amplification reaction is followed successively by 95 DEG C of pre-degeneration reaction 5min,
94 DEG C of reaction 30s, 58 DEG C of reaction 40s, 72 DEG C of reaction 40s, 35 cycles of progress, 10min is finally reacted at 72 DEG C, is obtained positive altogether
Control sample pcr amplification product.
4, electrophoresis detection
The blood serum sample pcr amplification product of gained, negative control sample pcr amplification product, positive control sample PCR are expanded
Volume increase object passes through 10gL-1Agarose gel electrophoresis carries out inspection diagnosis, specific as follows：
4.1 weigh 1.0g agaroses, are added in 100mL 1 × TAE buffer solutions, heating and melting in micro-wave oven, and 5 μ L are added
(100mg/mL) ethidium bromide, mixing are poured into horizontal positioned gel disk, and offset plate thickness is 5mm, is extracted after solidification to be cooled
Gel is put into electrophoresis tank by comb, and 1 × TAE buffer solutions are added and flood glue surface, obtain agarose buffer solution.
4.2 take 5 μ L blood serum samples pcr amplification products, negative control sample pcr amplification product, positive control sample respectively
Pcr amplification product and agarose buffer solution mixing are added in well, while adding 5 μ L DL2000DNA molecular weight standards.
4.3 voltage 80V~100V or electric current 40mA~50mA, electrophoresis 30min.
4.4 take out gel, are placed in uv analyzer and observe, or observe photograph in merging gel imaging system.
4.5 using DL2000DNA molecular masses standard as reference, and shaping band, positive control do not go out 260bp items to negative control
Band illustrates that control is set up；Occur 260bp bands in measuring samples pcr amplification product, as parrot beak ptilosis virus infects；It waits for
Sample product pcr amplification product is no band person, as negative.
Embodiment 4
1, the processing of whole blood sample
Wing venous blood sampling is carried out to suspected case parrot, anti-coagulants is added, obtains whole blood sample after mixing, it is standby
With.
2, DNA is extracted
2.1 whole blood sample DNA extractions：Supernatant merging 1.5mL sterile EP tubes obtained by 100 μ L are drawn, 800 μ L cracking are added
Liquid overturns mixing, stands cracking 10min, and the ice-cold absolute ethyl alcohols of 600 μ L are added, overturns mixing, staticly settles 10min,
12000r/min centrifuges 10min, discards supernatant liquid, is washed 1 time with 75% ethyl alcohol, discards ethyl alcohol, is inverted EP pipes, in air certainly
It is so dry, with 40 μ L 8mmolL-1NaOH dissolves, and obtains whole blood sample DNA extracting solutions.
2.2 negative control samples extract：100 μ L sterilizing tri-distilled water merging sterile centrifugation tubes are drawn, 800 μ L cracking are added
Liquid overturns mixing, stands cracking 10min, and the ice-cold absolute ethyl alcohols of 600 μ L are added, overturns mixing, staticly settles 10min,
12000r/min centrifuges 10min, discards supernatant liquid, is washed 1 time with 75% ethyl alcohol, discards ethyl alcohol, is inverted EP pipes, in air certainly
It is so dry, with 40 μ L 8mmolL-1NaOH dissolves, and obtains negative control sample extracting solution.
3, pcr amplification reaction
3.1 whole blood sample pcr amplification reactions：23 μ L of amplified reaction mixed liquor are taken, 2 μ L of whole blood sample DNA extracting solutions are added,
It is uniformly mixed, carries out pcr amplification reaction, the condition of pcr amplification reaction is followed successively by 95 DEG C of pre-degeneration reaction 5min, 94 DEG C of reactions
30s, 58 DEG C of reactions 40s, 72 DEG C of reaction 40s carry out 35 cycles altogether, and finally the reaction 10min at 72 DEG C, obtains whole blood sample PCR
Amplified production.
3.2 negative control sample pcr amplification reactions：23 μ L of amplified reaction mixed liquor are taken, negative control sample extraction is added
2 μ L of liquid are uniformly mixed, and carry out pcr amplification reaction, and the condition of pcr amplification reaction is followed successively by 95 DEG C of pre-degenerations reaction 5min, 94 DEG C
30s is reacted, 58 DEG C of reaction 40s, 72 DEG C of reaction 40s, 35 cycles of progress, 10min is finally reacted at 72 DEG C, obtains negative control altogether
Sample P CR amplified productions.
3.3 positive control sample pcr amplification reactions：23 μ L of amplified reaction mixed liquor are taken, parrot beak ptilosis virus sun is added
2 μ L of property grain are uniformly mixed, and carry out pcr amplification reaction, and the condition of pcr amplification reaction is followed successively by 95 DEG C of pre-degeneration reaction 5min,
94 DEG C of reaction 30s, 58 DEG C of reaction 40s, 72 DEG C of reaction 40s, 35 cycles of progress, 10min is finally reacted at 72 DEG C, is obtained positive altogether
Control sample pcr amplification product.
4, electrophoresis detection
The whole blood sample pcr amplification product of gained, negative control sample pcr amplification product, positive control sample PCR are expanded
Volume increase object passes through 10gL-1Agarose gel electrophoresis carries out inspection diagnosis, specific as follows：
4.1 weigh 1.0g agaroses, are added in 100mL 1 × TAE buffer solutions, heating and melting in micro-wave oven, and 5 μ L are added
(100mg/mL) ethidium bromide, mixing are poured into horizontal positioned gel disk, and offset plate thickness is 5mm, is extracted after solidification to be cooled
Gel is put into electrophoresis tank by comb, and 1 × TAE buffer solutions are added and flood glue surface, obtain agarose buffer solution.
4.2 take 5 μ L whole blood samples pcr amplification products, negative control sample pcr amplification product, positive control sample respectively
Pcr amplification product and agarose buffer solution mixing are added in well, while adding 5 μ L DL2000DNA molecular weight standards.
4.3 voltage 80V~100V or electric current 40mA~50mA, electrophoresis 30min.
4.4 take out gel, are placed in uv analyzer and observe, or observe photograph in merging gel imaging system.
4.5 using DL2000DNA molecular masses standard as reference, and shaping band, positive control do not go out 260bp items to negative control
Band illustrates that control is set up；Occur 260bp bands in measuring samples pcr amplification product, as parrot beak ptilosis virus infects；It waits for
Sample product pcr amplification product is no band person, as negative.
As a result, it has been found that suspected infection parrot beak ptilosis virus has 22 parts in 95 parts of clinical samples, positive rate is electrophoresis detection
23.1%.Wherein, Fig. 3 is the electrophoresis detection of partial clinical sample as a result, from the figure 3, it may be seen that 3/4/6 is parrot beak ptilosis virus sense
Contaminate positive findings；1/2/5/7/8/9 is negative findings.Field trial proves that the parrot beak ptilosis virus PCR of present invention gained is examined
Disconnected kit practicability and applicability are good.
Although the present invention is described in detail with a general description of the specific embodiments in this specification,
But on the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.
Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to claimed model
It encloses.
Claims (8)
1. a kind of parrot beak ptilosis virus PCR diagnostic kit, which is characterized in that including：Lysate, amplified reaction mixed liquor,
Negative control and positive control；Wherein, the ingredient of the lysate is DNAisoReagent；The amplified reaction mixed liquor packet
The tri-distilled water containing sterilizing, PCR Buffer, dNTP, PBFDV-F sense primer, PBFDV-R downstream primers and rTaq archaeal dna polymerases；
The negative control is sterilizing tri-distilled water；The positive control is PBFDV positive plasmids.
2. parrot beak ptilosis virus PCR diagnostic kit according to claim 1, which is characterized in that on the PBDFV-F
Trip primer sequence be：5’-AGCGATTTGGATAATGAGAAGTA-3’；The sequence of the PBDFV-R downstream primers is：5’-
CCCGGGGGGCCGTACACAACGT-3’。
3. parrot beak ptilosis virus PCR diagnostic kit according to claim 1, which is characterized in that the lysate at
It is divided into DNAiso Reagent, 20 reactions amount to 20mL, dress up 1 bottle；The amplified reaction mixed liquor includes：Sterilizing three is steamed
Water, 10 × PCR Buffer, 2.5mmolL-1dNTP、25μmol·L-1PBFDV-F sense primers, 25 μm of olL-1PBFDV-
R downstream primers and 5U/ μ L rTaq archaeal dna polymerases, each reaction volume ratio are 17.0 ︰, 2.5 ︰, 2 ︰, 0.5 ︰, 0.5 ︰ 0.5, amount to 23
μ L, 20 reactions amount to 460 μ L, dress up 1 pipe；The negative control is sterilizing tri-distilled water, and 20 reactions amount to 2.0mL, dress up 1
Bottle；The positive control is PBFDV positive plasmids, and 20 reactions amount to 40.0 μ L, dress up 1 pipe.
4. a kind of detection method of parrot beak ptilosis virus PCR diagnostic kit, which is characterized in that include the following steps：
Step 1, DNA is extracted
Sub-step 1.1, measuring samples DNA extractions：It draws measuring samples and is placed in sterile centrifugation tube, lysate is added, overturn mixing,
Cracking is stood, absolute ethyl alcohol is added, mixing is overturned, staticly settles, centrifuges, discards supernatant liquid, washs, discards cleaning solution, is inverted
Sterile centrifugation tube spontaneously dries in air, is dissolved with NaOH, obtains measuring samples DNA extracting solutions；
Sub-step 1.2, negative control sample extraction：It draws sterilizing tri-distilled water and is placed in sterile centrifugation tube, lysate is added, overturn mixed
It is even, cracking is stood, absolute ethyl alcohol is added, mixing is overturned, staticly settles, centrifuges, discards supernatant liquid, washs, discards cleaning solution,
Sterile centrifugation tube is set, is spontaneously dried in air, is dissolved with NaOH, negative control sample extracting solution is obtained；
Step 2, pcr amplification reaction
Sub-step 2.1, measuring samples pcr amplification reaction：Amplified reaction mixed liquor is taken, the measuring samples DNA extracting solutions are added,
Be uniformly mixed, carry out pcr amplification reaction, the condition of the pcr amplification reaction be followed successively by 95 DEG C of pre-degenerations reaction 5min, 94 DEG C it is anti-
30s is answered, 58 DEG C of reaction 40s, 72 DEG C are reacted 40s, carry out multiple cycles altogether, are finally reacted 10min at 72 DEG C again, are obtained measuring samples
Pcr amplification product；
Sub-step 2.2, negative control sample pcr amplification reaction：Amplified reaction mixed liquor is taken, the negative control sample is added and carries
Take liquid, be uniformly mixed, carry out pcr amplification reaction, the condition of the pcr amplification reaction be followed successively by 95 DEG C of pre-degenerations reaction 5min,
94 DEG C of reaction 30s, 58 DEG C of reaction 40s, 72 DEG C are reacted 40s, carry out multiple cycles altogether, are finally reacted 10min at 72 DEG C again, are obtained cloudy
Property control sample pcr amplification product；
Sub-step 2.3, positive control sample pcr amplification reaction：Amplified reaction mixed liquor is taken, parrot beak ptilosis virus-positive is added
Plasmid is uniformly mixed, and carries out pcr amplification reaction, the condition of the pcr amplification reaction be followed successively by 95 DEG C of pre-degenerations reaction 5min,
94 DEG C of reactions 30s, 58 DEG C of reactions 40s, 72 DEG C of reaction 40s carry out multiple cycles altogether, and finally the reaction 10min at 72 DEG C, obtains positive
Control sample pcr amplification product；
Step 3, electrophoresis detection
By agarose gel electrophoresis respectively to the measuring samples pcr amplification product, negative control sample pcr amplification product and
Positive control sample pcr amplification product carries out electrophoresis detection, and is judged.
5. the detection method of parrot beak ptilosis virus PCR diagnostic kit according to claim 4, which is characterized in that step
In rapid 2, the amplified reaction mixed liquor includes sterilizing tri-distilled water, PCR Buffer, dNTP, PBFDV-F sense primer, PBFDV-
R downstream primers and rTaq archaeal dna polymerases.
6. the detection method of parrot beak ptilosis virus PCR diagnostic kit according to claim 5, which is characterized in that institute
The sequence for stating PBDFV-F sense primers is：5’-AGCGATTTGGATAATGAGAAGTA-3’；The PBDFV-R downstream primers
Sequence is：5’-CCCGGGGGGCCGTACACAACGT-3’.
7. the detection method of parrot beak ptilosis virus PCR diagnostic kit according to claim 4, which is characterized in that step
In rapid 2, the multiple cycle is 35 cycles.
8. the detection method of parrot beak ptilosis virus PCR diagnostic kit according to claim 4, which is characterized in that step
In rapid 3, the standard of the judgement is：Negative control sample pcr amplification product not produce by shaping band, positive control sample PCR amplification
Object goes out 260bp bands, illustrates that control is set up；Occur 260bp bands, as parrot beak ptilosis in measuring samples pcr amplification product
Virus infection；Measuring samples pcr amplification product is no band person, as negative.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810359781.1A CN108588278A (en) | 2018-04-20 | 2018-04-20 | A kind of parrot beak ptilosis virus PCR diagnostic kit and its detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810359781.1A CN108588278A (en) | 2018-04-20 | 2018-04-20 | A kind of parrot beak ptilosis virus PCR diagnostic kit and its detection method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108588278A true CN108588278A (en) | 2018-09-28 |
Family
ID=63614273
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810359781.1A Pending CN108588278A (en) | 2018-04-20 | 2018-04-20 | A kind of parrot beak ptilosis virus PCR diagnostic kit and its detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108588278A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110607396A (en) * | 2019-08-21 | 2019-12-24 | 青岛立见诊断技术发展中心 | Parrot multiple virus detection primer, detection kit, virus detection method and application |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE10344545B4 (en) * | 2003-09-19 | 2005-12-22 | Universität Leipzig | Genetically modified capsid protein of the Beak and Feather Disease Virus |
| CN103038343A (en) * | 2010-03-23 | 2013-04-10 | 英特瑞克斯顿股份有限公司 | Vectors conditionally expressing therapeutic proteins, host cells comprising the vectors, and uses thereof. |
| US20130267429A1 (en) * | 2009-12-21 | 2013-10-10 | Lawrence Livermore National Security, Llc | Biological sample target classification, detection and selection methods, and related arrays and oligonucleotide probes |
| CN104561381A (en) * | 2015-01-10 | 2015-04-29 | 福建省农业科学院畜牧兽医研究所 | Primers and probe for detecting pigeon circovirus |
| CN107586887A (en) * | 2017-10-31 | 2018-01-16 | 咸阳职业技术学院 | A kind of porcine circovirus 2 type and 3 type PCR differential diagnosis kits and its detection method |
-
2018
- 2018-04-20 CN CN201810359781.1A patent/CN108588278A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE10344545B4 (en) * | 2003-09-19 | 2005-12-22 | Universität Leipzig | Genetically modified capsid protein of the Beak and Feather Disease Virus |
| US20130267429A1 (en) * | 2009-12-21 | 2013-10-10 | Lawrence Livermore National Security, Llc | Biological sample target classification, detection and selection methods, and related arrays and oligonucleotide probes |
| CN103038343A (en) * | 2010-03-23 | 2013-04-10 | 英特瑞克斯顿股份有限公司 | Vectors conditionally expressing therapeutic proteins, host cells comprising the vectors, and uses thereof. |
| CN104561381A (en) * | 2015-01-10 | 2015-04-29 | 福建省农业科学院畜牧兽医研究所 | Primers and probe for detecting pigeon circovirus |
| CN107586887A (en) * | 2017-10-31 | 2018-01-16 | 咸阳职业技术学院 | A kind of porcine circovirus 2 type and 3 type PCR differential diagnosis kits and its detection method |
Non-Patent Citations (4)
| Title |
|---|
| HADDADMARANDI等: ""Molecular detection and characterization of beak and feather disease virus in psittacine birds in Tehran, Iran"", 《IRANIAN JOURNAL OF VETERINARY RESEARCH》 * |
| SHUBHAGATA DAS等: ""A comparison of PCR assays for beak and feather disease virus and highresolution melt (HRM) curve analysis of replicase associated proteinand capsid genes"", 《JOURNAL OF VIROLOGICAL METHODS》 * |
| 庄青叶等: ""我国大陆地区首例鹦鹉喙羽病的诊断及其致病病毒C1基因的序列分析"", 《畜牧与兽医》 * |
| 徐云碧等: "《分子数量遗传学》", 31 December 1994, 中国农业出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110607396A (en) * | 2019-08-21 | 2019-12-24 | 青岛立见诊断技术发展中心 | Parrot multiple virus detection primer, detection kit, virus detection method and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Deng et al. | Diagnosis of Porcine teschovirus encephalomyelitis in the Republic of Haiti | |
| CN107586887B (en) | PCR differential diagnosis kit for porcine circovirus type 2 and type 3 and detection method thereof | |
| CN108315490A (en) | Pig acute diarrhea syndrome coronavirus and Delta coronavirus RT-PCR diagnostic kits and its detection method | |
| CN108085416A (en) | A kind of fowl neoplastic disease triple fluorescent PCR detection kit and its detection method | |
| CN103131798A (en) | Norovirus real-time fluorescent RT-PCR detection kit and application thereof | |
| CN107012261A (en) | Ana 1 aviadenovirus A types and the dual EvaGreen real-time fluorescence quantitative PCRs detection primer of 2 types | |
| Negash et al. | Molecular evidence of very virulent infectious bursal disease viruses in chickens in Ethiopia | |
| CN106399585A (en) | Universal PCR primers and method for detecting group I aviadenovirus and detection kit | |
| CN107619822A (en) | A kind of pig epidemic diarrhea virus attenuated strain, its cultural method and application | |
| CN113832260A (en) | Goose astrovirus, goose parvovirus and goose calicivirus multiplex nano PCR (polymerase chain reaction) detection primer pair, kit and application method | |
| CN103627818B (en) | The LAMP kit of detection Main Subtype avian leukosis virus | |
| CN105671169B (en) | Citrus Huanglongbing pathogen bacterium Asia kind detection primer, kit and detection method | |
| CN106435031A (en) | PCR detection kit for specific detection of fowl adenovirus group I and detection method thereof | |
| Bala et al. | Identification of strain diversity and phylogenetic analysis based on two major essential proteins of Orf viruses isolated from several clinical cases reported in Malaysia | |
| CN108588278A (en) | A kind of parrot beak ptilosis virus PCR diagnostic kit and its detection method | |
| CN106435020B (en) | For detecting the universal kit of different genotype infectious bronchitis virus | |
| CN117050989A (en) | Primer probe group for identifying spike messenger scorpion and application thereof | |
| CN111154915A (en) | PCR differential diagnosis kit for porcine circovirus type 4 and type 3 and detection method thereof | |
| CN108315492A (en) | A kind of birds polyomavirus PCR diagnostic kits and its detection method | |
| CN107298700A (en) | Artificial reconstructed PCV2 Rep albumen, restructuring PCV2 viruses and its application | |
| Zhu et al. | Identification and rapid diagnosis of the pathogen responsible for haemorrhagic disease of the gill of Allogynogenetic crucian carp | |
| CN105256071A (en) | Method for quickly detecting carp herpes virus type III | |
| CN106521037B (en) | One kind is for diagnosing FAdV/MDV/ALV/REV4 weight PCR detection kit | |
| CN113564132B (en) | Coxsackie virus A16 type strain and application thereof | |
| CN109439799A (en) | It is a kind of for detecting the sleeve type PCR primer and kit of pig acute diarrhea coronavirus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180928 |